Protein L-Isoaspartyl Methyltransferase Catalyzes in Vivo Racemization of Aspartate-25 in Mammalian Histone H2B

Glen W Young,Sarah A Hoofring,Mark J Mamula,Hester A Doyle,Gerard J Bunick,Yonglin Hu,Dana W Aswad

doi:10.1074/jbc.m503624200

Abstract

Protein L-isoaspartyl methyltransferase (PIMT) has been implicated in the repair or metabolism of proteins containing atypical L-isoaspartyl peptide bonds. The repair hypothesis is supported by previous studies demonstrating in vitro repair of isoaspartyl peptides via formation of a succinimide intermediate. Utilization of this mechanism in vivo predicts that PIMT modification sites should exhibit significant racemization as a side reaction to the main repair pathway. We therefore studied the D/L ratio of aspartic acid at specific sites in histone H2B, a known target of PIMT in vivo. Using H2B from canine brain, we found that Asp25 (the major PIMT target site in H2B) was significantly racemized, exhibiting d/l ratios as high as 0.12, whereas Asp51, a comparison site, exhibited negligible racemization (D/L < or = 0.01). Racemization of Asp25 was independent of animal age over the range of 2-15 years. Using H2B from 2-3-week mouse brain, we found a similar D/L ratio (0.14) at Asp25 in wild type mice, but substantially less racemization (D/L = 0.035) at Asp25 in PIMT-deficient mice. These findings suggest that PIMT functions in the repair, rather than the metabolic turnover, of isoaspartyl proteins in vivo. Because PIMT has numerous substrates in cells, these findings also suggest that D-aspartate may be more common in cellular proteins than hitherto imagined and that its occurrence, in some proteins at least, is independent of animal age.

Highlights

Protein L-isoaspartyl methyltransferase (PIMT)1 selectively methylates atypical L-isoaspartyl sites in proteins [1,2,3,4]
Further evidence that PIMT serves to repair L-isoaspartyl sites in proteins comes from two lines of study in which PIMT activity was artificially reduced in living cells
Using rat PC12 cells, we previously estimated that isoaspartyl sites are generated in roughly 1% of H2B molecules/day in vivo [20]

Summary

Introduction

Protein L-isoaspartyl methyltransferase (PIMT) selectively methylates atypical L-isoaspartyl sites in proteins [1,2,3,4]. A repair function is supported by in vitro studies using PIMT and defined polypeptide substrates [13,14,15] A second line of study subsequently demonstrated that proteins in tissue extracts of PIMT-deficient (knock-out) mice show dramatically higher levels of isoaspartate than do wild type mice [18, 19] Both of these observations are consistent with a repair role for PIMT, both are consistent with a possible role for PIMT in facilitating the degradation of isoaspartyl proteins. On the other hand, PIMT-dependent methylation functions to target isoaspartyl proteins to a degradation pathway ( avoiding repeated cycles of succinimide formation), we would not expect increased racemization at the PIMT target site in either normal or PIMT-deficient animals

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Results

Conclusion